CN103589756A - Method for biosynthesizing S-1, 2-propylene glycol by using L-lactic acid - Google Patents
Method for biosynthesizing S-1, 2-propylene glycol by using L-lactic acid Download PDFInfo
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Abstract
The invention discloses a method for biosynthesizing S-1, 2-propylene glycol by using L-lactic acid. The method specifically comprises the steps as follows: L-lactic acid or salt of L-lactic acid is converted into the S-1, 2-propylene glycol through a recombination biological cell; the recombination biological cell is provided with an enzyme system with a function of converting L-lactic acid or salt of L-lactic acid into the S-1, 2-propylene glycol; and the enzyme system comprises (a1), (a2) and (a3), wherein the (a1) has an enzyme 1 which has a function of converting L-lactic acid or salt of L-lactic acid into an L-lactyl coenzyme A, the (a2) has an enzyme 2 which has a function of converting the L-lactyl coenzyme A into L-lactaldehyde, and the (a3) has an enzyme 3 which has a function of converting the L-lactaldehyde into the S-1, 2-propylene glycol. The method has important significance to establishment of an S-1, 2-propylene glycol biosynthesizing method which has independent property right, lower material cost, brand new synthetic route and higher conversion efficiency.
Description
Technical field
The present invention relates to a kind of S-1 of preparation, the method for 2-propylene glycol, particularly a kind of recombination bacillus coli that utilizes is converted into S-1 by Pfansteihl and salt form thereof, the method for 2-propylene glycol.
Background technology
The structure of propylene glycol is divided into 1,2-PD and 1,3-PD.1,2-PD is mainly used to produce unsaturated polyester resin, frostproofer, softening agent and substitutes ethylene glycol and make refrigerant and thermal barrier etc. for antifreeze aircraft and at food, and it also can be used for nonionic detergent or wetting Agent for Printing Inks.Propylene glycol or good solvent, can be used for food seasoning and spices, or the ointment ointment of medicine industry etc.
About the synthetic R-1 of biocatalysis, the research of 2-propylene glycol, at present, external existing about the synthetic R-1 of biocatalysis, the report of 2-propylene glycol, Tadashi etc. adopt airlift fermentor to generate R-1 to yeast reduction hydroxyacetone, 2-propylene glycol and yeast oxidation resolution of racemic 1,2-propylene glycol is studied, but obtains industrial applications not yet, and domestic rare report.
Summary of the invention
The object of this invention is to provide a kind of S-1 of preparation, the method for 2-propylene glycol, is specially Pfansteihl biosynthesizing S-1, the method for 2-propylene glycol utilized.
A kind of S-1 for preparing provided by the present invention, the method for 2-propylene glycol, comprises with restructuring biomass cells and Pfansteihl or its salt is converted into S-1, the step of 2-propylene glycol; Described restructuring biomass cells is expressed to have and Pfansteihl or its salt is converted into S-1, the enzyme system of 2-propylene glycol function.
In aforesaid method, described Pfansteihl or its salt are converted into S-1,2-propylene glycol, specifically comprises the step of following (1)-(3):
(1) Pfansteihl or its salt are converted into L-lactoyl coenzyme A;
(2) described L-lactoyl coenzyme A is converted into L-lactic aldehyde;
(3) described L-lactic aldehyde is converted into S-12-propylene glycol.
In aforesaid method, described enzyme system specifically comprises as follows (a1)-(a3):
(a1) there is the enzyme 1 that Pfansteihl or its salt is converted into L-lactoyl coenzyme A function;
(a2) there is the enzyme 2 that described L-lactoyl coenzyme A is converted into L-lactic aldehyde function;
(a3) have described L-lactic aldehyde is converted into S-1, the enzyme 3 of 2-propylene glycol function.
In the present invention, described enzyme 1 is propionyl-coa transferase; Described enzyme 2 is coenzyme A dependent form succinic semialdehyde dehydrogenase; Described enzyme 3 is 3-hydroxymalonate dehydrogenase.
Further, described propionyl-coa transferase is the point mutation albumen of the propionyl-coa transferase of clostridium propionicum (Clostridium propionicum), and its aminoacid sequence is specifically as shown in sequence in sequence table 1; Described coenzyme A dependent form succinic semialdehyde dehydrogenase is the coenzyme A dependent form succinic semialdehyde dehydrogenase of Yersinia enterocolitica (Yersinia enterocolitica), and its aminoacid sequence is specifically as shown in sequence in sequence table 2; Described 3-hydroxymalonate dehydrogenase is the 3-hydroxymalonate dehydrogenase of bacillus cereus (Bacillus cereus), and its aminoacid sequence is specifically as shown in sequence in sequence table 3.
Wherein, sequence 1 is comprised of 524 amino acid; Sequence 2 is comprised of 462 amino acid; Sequence 3 is comprised of 292 amino acid.
Described restructuring biomass cells is following encoding gene to be imported to the restructuring biomass cells of the described enzyme system of expression obtaining after object biomass cells:
The encoding gene of propionyl-coa transferase as described in as shown in sequence in sequence table 4;
The encoding gene of coenzyme A dependent form succinic semialdehyde dehydrogenase as described in as shown in sequence in sequence table 5;
The encoding gene of 3-hydroxymalonate dehydrogenase as described in as shown in sequence in sequence table 6.
Wherein, sequence 4 is comprised of 1575 Nucleotide, the albumen shown in sequence 1 in code sequence list; Sequence 5 is comprised of 1389 Nucleotide, the albumen shown in sequence 2 in code sequence list; Sequence 6 is comprised of 879 Nucleotide, the albumen shown in sequence 3 in code sequence list.
In one embodiment of the invention, the encoding gene of described enzyme system is imported to object biomass cells, be specially by the recombinant plasmid carrying and express the encoding gene of described enzyme system imported and realize in described object biomass cells.Described recombinant plasmid is specially following recombinant plasmid A and following recombinant plasmid B.
In aforesaid method, described biomass cells can be microorganism cells, zooblast or vegetable cell.
In one embodiment of the invention, described biomass cells is specially intestinal bacteria, as intestinal bacteria Origami(DE3).
Further, described restructuring biomass cells is following recombination bacillus coli 1.
In one embodiment of the invention, in described method, described " with restructuring biomass cells, Pfansteihl or its salt being converted into S-1,2-propylene glycol " is specially with described restructuring biomass cells and Pfansteihl sodium is converted into S-1,2-propylene glycol.
In described method, described " Pfansteihl or its salt (as Pfansteihl sodium) being converted into S-12-propylene glycol with restructuring biomass cells " specifically comprises following (A) and step (B):
(A) to adding final concentration in the nutrient solution of the described recombination bacillus coli 1 of OD600=0.6, be 0.2%(0.2g/100mL) L-arabinose, 37 ℃ of 180rpm concussions are cultivated, after 1h, adding final concentration is the IPTG of 0.05mM, goes to 20 ℃ of 180rpm concussions and cultivates 16-18h;
(B) collect thalline, by concentration, be first 0.9%(0.9g/100mL) the NaCl aqueous solution wash twice, after use 100mM phosphate buffered saline buffer (pH7.4) resuspended to OD600=30, adding final concentration is Pfansteihl or its salt (as Pfansteihl sodium) of 100mM, 37 ℃, 200rpm, cultivates 48h, from nutrient solution, obtain S-1,2-propylene glycol.
A further object of the present invention is to provide (I) or biomaterial (II) or (III) as follows.
(I) all or part of in following DNA fragmentation:
(b1) nucleotides sequence is classified the DNA fragmentation shown in sequence 4 in sequence table (nucleotide sequence after the point mutation optimization of the propionyl-coa transferase of clostridium propionicum (Clostridium propionicum)) as;
(b2) nucleotides sequence is classified the DNA fragmentation shown in sequence 5 in sequence table (nucleotide sequence after the optimization of the coenzyme A dependent form succinic semialdehyde dehydrogenase of Yersinia enterocolitica (Yersinia enterocolitica)) as;
(b3) nucleotides sequence is classified the DNA fragmentation shown in sequence 6 in sequence table (nucleotide sequence after the optimization of the 3-hydroxymalonate dehydrogenase of bacillus cereus (Bacillus cereus)) as;
The above is optimized under the prerequisite of aminoacid sequence that does not change corresponding enzyme, the codon of wild type gene is replaced with to the codon of intestinal bacteria preference (high frequency use).
(II) recombinant plasmid A and/or recombinant plasmid B:
Described recombinant plasmid A is for carrying and express the recombinant expression vector of DNA fragmentation described in (b1); Described recombinant plasmid B for carry and express DNA fragmentation described in (b2) and (b3) described in the recombinant expression vector of DNA fragmentation.
In described recombinant plasmid A, starting the promotor that described in (b1), DNA fragmentation is transcribed is araBAD promotor; In described recombinant plasmid B, start DNA fragmentation described in (b2) and (b3) described in the DNA fragmentation promotor of transcribing be T7 promotor.
Concrete, described recombinant plasmid A is at the recombinant plasmid (pBAD-pct) obtaining after DNA fragmentation as shown in sequence 4 during the multiple clone site (as EcoR I and Hind III) of pBAD43 carrier is located insertion sequence table; Described recombinant plasmid B at the multiple clone site 1(of pETDUET-1 carrier as Nco I and Hind III) locate insertion sequence table in DNA fragmentation as shown in sequence 5, simultaneously at multiple clone site 2(as Nde I and Xho I) recombinant plasmid (pETDUET-pm) that obtains after DNA fragmentation as shown in sequence 6 in locating insertion sequence table.
(III) recombination bacillus coli 1 or/and recombination bacillus coli 2 or/and recombination bacillus coli 3:
Described recombination bacillus coli 1 is for carrying recombinant plasmid A described in (II) and described recombinant plasmid B, and express DNA fragmentation described in DNA fragmentation, (b2) described in (b1) and (b3) described in the intestinal bacteria of DNA fragmentation;
Described recombination bacillus coli 2 is for carrying recombinant plasmid A described in (II), and the intestinal bacteria of DNA fragmentation described in expression (b1);
Described recombination bacillus coli 3 is for carrying recombinant plasmid B described in (II), and expresses b2) described in DNA fragmentation and (b3) described in the intestinal bacteria of DNA fragmentation.
In one embodiment of the invention, described recombination bacillus coli 1 is for adopting described recombinant plasmid A and described recombinant plasmid B cotransformation intestinal bacteria Origami(DE3) after, the intestinal bacteria of the described enzyme 1-of the expression enzyme 3 obtaining.
Certainly; so long as adopt the Pfansteihl biosynthesizing S-1 that utilizes provided by the present invention; the three-step reaction of 2-propylene glycol; or some step reactions wherein; synthesize S-1,2-propylene glycol, contains (b1)-(b3) described DNA fragmentation; or the expression cassette of some DNA fragmentations wherein, recombinant cell lines, and other recombinant bacteriums except above-mentioned recombination bacillus coli also all belong to protection scope of the present invention.
Described biomaterial is utilizing Pfansteihl or its salt (as Pfansteihl sodium) to synthesize S-1, and the application in 2-propylene glycol also belongs to protection domain of the present invention.
The present invention has set up that brand-new to take Pfansteihl or its salt (as Pfansteihl sodium) be raw material, prepares S-1, the method for 2-propylene glycol by biosynthetic pathway.The present invention has autonomous property right, cost of material is cheap, route of synthesis is brand-new, transformation efficiency is higher S-1 to setting up, and 2-propylene glycol biosynthetic means is significant.
Accompanying drawing explanation
Fig. 1 is that the enzyme of recombinant expression vector pBAD-pct, middle interstitial granules pETDUET-pdcd and recombinant expression vector pETDUET-pm is cut evaluation collection of illustrative plates.Wherein, swimming lane M is DNA molecular amount standard; Swimming lane 1 and 3 is the BamH I single endonuclease digestion result of recombinant expression vector pBAD-pct; The recombinant plasmid pBAD-pct contrast of swimming lane 2 and 4 for cutting without enzyme; Swimming lane 5 and 6 is the Sph I single endonuclease digestion result of middle interstitial granules pETDUET-pdcd; Swimming lane 7 and 8 is the BsaA I single endonuclease digestion result of recombinant expression vector pETDUET-pm.
Fig. 2 is S-1,2-propylene glycol standard substance and recombinant bacterial strain Origami(DE3) the HPLC collection of illustrative plates of the HPLC collection of illustrative plates of the ferment filtrate of/pBAD-pct/pETDUET-pm.Wherein, a) be S-1, the HPLC collection of illustrative plates of 2-propylene glycol standard substance; B) be recombinant bacterial strain Origami(DE3) the HPLC collection of illustrative plates of the ferment filtrate of/pBAD-pct/pETDUET-pm.
Fig. 3 is S-1,2-propylene glycol standard substance and recombinant bacterial strain Origami(DE3) the Mass Spectrometric Identification result collection of illustrative plates of the ferment filtrate of/pBAD-pct/pETDUET-pm.Wherein, A is S-1,2-propylene glycol standard substance; B is recombinant bacterial strain Origami(DE3) ferment filtrate of/pBAD-pct/pETDUET-pm by ethyl acetate extraction after sample
Embodiment
In the present invention, prepare S-1, the method for 2-propylene glycol, is by enzyme system, Pfansteihl or its salt (as Pfansteihl sodium) to be converted into S-1,2-propylene glycol in biomass cells.Specifically comprise following three steps:
(1) Pfansteihl or its salt are converted into L-lactoyl coenzyme A;
(2) described L-lactoyl coenzyme A is converted into L-lactic aldehyde;
(3) described L-lactic aldehyde is converted into S-1,2-propylene glycol.
Above 6 steps reactions in turn corresponding enzyme be (a1)-(a3) as follows:
(a1) there is the enzyme 1 that Pfansteihl or its salt is converted into L-lactoyl coenzyme A function, as propionyl-coa transferase;
(a2) there is the enzyme 2 that described L-lactoyl coenzyme A is converted into L-lactic aldehyde function, as coenzyme A dependent form succinic semialdehyde dehydrogenase;
(a3) have described L-lactic aldehyde is converted into S-1, the enzyme 3 of 2-propylene glycol function, as 3-hydroxymalonate dehydrogenase.
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
PBAD43 carrier: Addgene company, catalog number is VYA0258.
PETDUET-1 carrier: Novagen company, catalog number is 71146.
Intestinal bacteria Origami(DE3): Novagen company, catalog number is 70627.
One, the structure of recombinant expression vector pBAD-pct and pETDUET-pm
1, selection and the optimization of step (1) enzyme gene used in 3 step catalyzed reactions
Catalyzed reaction for step (1) in 3 step reactions, choose the CDS complete sequence (the 485-2059 position of GenBank:AJ276553.1) of gene (pct) of the propionyl-coa transferase of clostridium propionicum (Clostridium propionicum), through designing and repeatedly verifying and obtain being suitable for the Optimization-type propionyl-coa transferase gene order at expression in escherichia coli, be about to the codon optimized sequence that wild-type propionyl-coa transferase gene order is transformed into intestinal bacteria preference (high frequency use) under the prerequisite that does not change aminoacid sequence, thereby improve the expression level of propionyl-coa transferase in intestinal bacteria culture environment.
Then, according to pertinent literature report (Taek Ho Yang, Tae Wan Kim, Hye Ok Kang, Sang-Hyun Lee, Eun Jeong Lee, Sung-Chul Lim, Sun Ok Oh, Ae-Jin Song, Si Jae Park, Sang Yup Lee.Biosynthesis of polylactic acid and its copolymers using evolved propionate CoA transferase and PHA synthase.Biotechnology and Bioengineering, 2010, 105, 150-160.), the codon of the coding Ala of intestinal bacteria preference (high frequency use) will be changed into corresponding to three Nucleotide of the 193rd Val of propionyl-coa transferase Argine Monohydrochloride sequence in the Optimization-type propionyl-coa transferase gene order of above gained, be intended to reduce in protein expression process foreign protein to the restraining effect of host e. coli (reference " Taek Ho Yang, Tae Wan Kim, Hye Ok Kang, Sang-Hyun Lee, Eun Jeong Lee, Sung-Chul Lim, Sun Ok Oh, Ae-Jin Song, Si Jae Park, Sang Yup Lee.Biosynthesis of polylactic acid and its copolymers using evolved propionate CoA transferase and PHA synthase.Biotechnology and Bioengineering, 2010, 105, 150-160. " in have report).
According to aforesaid method, final acquisition by the Optimization-type point mutation propionyl-coa transferase encoding gene of intestinal bacteria preference design, its gene order is sequence 4 in sequence table, consists of altogether 1575 Nucleotide.The albumen of this genes encoding is the albumen (being point mutation propionyl-coa transferase) that in sequence table, aminoacid sequence shown in sequence 1 forms, compare with the albumen of wild-type propionyl-coa transferase gene (the 485-2059 position of GenBank:AJ276553.1) coding, wherein the Val of the 193rd sports Ala(V193A).
For the catalyzed reaction of step (2), choose the gene (pcdD) of the coenzyme A dependent form succinic semialdehyde dehydrogenase of Yersinia enterocolitica (Yersinia enterocolitica), to it, adopt the same mode to carry out codon optimized.The nucleotide sequence of the described coenzyme A dependent form succinic semialdehyde dehydrogenase gene after optimization, as shown in sequence in sequence table 5, is comprised of 1388 Nucleotide altogether.Protein shown in sequence 2 in this gene coded sequence table.
For the catalyzed reaction of step (3), choose the gene (mmsB) of the 3-hydroxymalonate dehydrogenase of bacillus cereus (Bacillus cereus), to it, adopt the same mode to carry out codon optimized.The nucleotide sequence of the described 3-hydroxymalonate dehydrogenase gene after optimization, as shown in sequence in sequence table 6, is comprised of 879 Nucleotide altogether.Protein shown in sequence 3 in this gene coded sequence table.
2, the structure of recombinant expression vector pBAD-pct
The DNA fragmentation shown in sequence 4 in sequence table of take is template, with primer 1 and primer 2, carries out pcr amplification.
Primer 1:5 '-GAG
gAATTCaTGCGCAAAGTTCCGATTA-3 ' (underscore is partly the recognition sequence of restriction enzyme EcoR I, and sequence is thereafter the 1-19 position of sequence 4);
Primer 2: 5 '-CCGG
aAGCTTaGCTTTTCATTTCTTTCAGG-3 ' (underscore is partly the recognition sequence of restriction enzyme Hind III, and the 9-30 position of this sequence is the reverse complementary sequence of the 1554-1575 position of sequence 4).
With restriction enzyme EcoR I and Hind III double digestion pcr amplification product, after reclaiming, glue is connected with the pBAD43 carrier framework large fragment through same double digestion, obtain recombinant plasmid.Will be through the recombinant plasmid sample presentation order-checking of restriction enzyme BamH I single endonuclease digestion preliminary evaluation correct (object band 752bp and 6956bp, the swimming lane 1 and 3 in Fig. 1).To show the recombinant plasmid called after pBAD-pct obtaining after DNA fragmentation shown in sequence 4 in insertion sequence table between the multiple clone site EcoR of pBAD43 carrier I and Hind III through order-checking.In recombinant expression vector pBAD-pct, the promotor that shown in initiating sequence 4, DNA fragmentation is transcribed is araBAD promotor.
3, the structure of recombinant expression vector pETDUET-pm
The DNA fragmentation shown in sequence 5 in sequence table of take is template, with primer 3 and primer 4, carries out pcr amplification, obtains PCR product 1.The DNA fragmentation shown in sequence 6 in sequence table of take is template, with primer 5 and primer 6, carries out pcr amplification, obtains PCR product 2.
Primer 3:5 '-GAG
tCATGAaCACCAACGATCTGGAATC-3 ' (underscore is partly the recognition sequence of restriction enzyme BspH I, and the 6-28 position of this sequence is the 1-23 position of sequence 5);
Primer 4:5 '-CGC
aAGCTTaACGGATAGAAAAACCATT-3 ' (underscore is partly the recognition sequence of restriction enzyme Hind III, and the 8-28 of this sequence is the reverse complementary sequence of the 1369-1389 position of sequence 5).
Primer 5:5 '-CGCG
cTAGaTGGAACATAAAACTTTATCA-3 ' (underscore is partly the recognition sequence of restriction enzyme Bfa I, and sequence is thereafter the 1-21 position of sequence 6);
Primer 6:5 '-GCG
cTCGAGtTACCCCCTTATATATTTT-3 ' (underscore is partly the recognition sequence of restriction enzyme Xho I, and sequence is thereafter the reverse complementary sequence of the 861-879 position of sequence 6).
With restriction enzyme BspH I(and Nco I, be isocaudarner) and Hind III double digestion PCR product 1, after reclaiming, glue is connected with the pETDUET-1 carrier framework large fragment through Nco I and Hind III double digestion, obtain middle interstitial granules pETDUET-pdcd.By restriction enzyme Sph I single endonuclease digestion for middle interstitial granules pETDUET-pdcd, result obtains two object bands (swimming lane 5 in Fig. 1 and swimming lane 6) that size is about 1222bp and 5513bp, consistent with expected results, in proving, interstitial granules pETDUET-pdcd builds correct.
Using restriction enzyme Bfa I and Xho I double digestion PCR product 2, is isocaudarner with process Nde I(with Bfa I after glue reclaims again) be connected with the middle interstitial granules pETDUET-pdcd skeleton large fragment of Xho I double digestion, obtain recombinant plasmid.By restriction enzyme BsaA I single endonuclease digestion for recombinant plasmid, result obtains three object bands (swimming lane 7 in Fig. 1 and swimming lane 8) that size is about 1708bp, 2270bp and 3584bp, consistent with expected results.
To through enzyme, cut and identify that tentative confirmation builds correct recombinant plasmid sample presentation order-checking above, by through order-checking, show the multiple clone site Nco of pETDUET-1 carrier I and Hind III and between DNA fragmentation shown in sequence 5 in insertion sequence table, recombinant plasmid called after pETDUET-pm simultaneously that obtain after DNA fragmentation shown in sequence 6 in insertion sequence table between multiple clone site Nde I and Xho I.In recombinant expression vector pETDUET-pm, the promotor that shown in the promotor that shown in initiating sequence 5, DNA fragmentation is transcribed and initiating sequence 6, DNA fragmentation is transcribed is T7 promotor.
Two, for synthesizing S-1, the structure of the recombination bacillus coli of 2-propylene glycol from Pfansteihl or its salt
Recombinant expression vector pBAD-pct in step 1 and pETDUET-pm cotransformation are entered to intestinal bacteria Origami(DE3) in, after conversion, be applied on the LB solid medium flat board that contains 100 μ g/mL ammonia benzyl mycins and 100 μ g/mL spectinomycins and carry out pressurized screen choosing, several single bacterium colonies of picking, be inoculated in the LB liquid nutrient medium containing 100 μ g/mL ammonia benzyl mycins and 100 μ g/mL spectinomycins 37 ℃ of thermal agitation overnight incubation.Take bacterium liquid as template, use respectively primer pair 1(primer 1/ primer 2), primer pair 2(primer 3/ primer 4), primer pair 3(primer 5/ primer 6) carry out pcr amplification, can increase respectively and obtain the positive recombinant bacterial strain of mono-clonal bacterial strain that size is about the object band of 1580bp, 1396bp and 885bp, called after Origami(DE3)/pBAD-pct/pETDUET-pm.Experiment arrange simultaneously pBAD43 empty carrier and pETDUET-1 empty carrier cotransformation are entered to intestinal bacteria Origami(DE3) contrast, gained recombinant bacterial strain called after Origami(DE3)/pBAD43/pETDUET-1.
One, by biological fermentation, Pfansteihl sodium is converted into S-12-propylene glycol
The recombination bacillus coli Origami(DE3 that embodiment 1 is built)/pBAD-pct/pETDUET-pm incubated overnight, obtains seed liquor, and 1% ratio is inoculated in new LB substratum by volume, and 37 ℃ of 180rpm concussions are cultivated.When OD600 to 0.6 left and right, adding final concentration is 0.2%(0.2g/100mL) L-arabinose, after 1h, adding final concentration is that the IPTG of 0.05mM induces, and goes to 20 ℃ of 180rpm concussions and cultivates 16-18h.
Induction is received bacterium later, and 4 ℃, 5000rpm, centrifugal 10min.After use 0.9%(0.9g/100mL) NaCl washes twice.After use that 100mM phosphate buffered saline buffer (pH7.4) is resuspended is about 30 left and right to OD600.Add final concentration be the Pfansteihl sodium of 100mM as substrate, 37 ℃, full cell response is carried out in 200rpm reaction for 24 hours.Afterwards, get fermented liquid, for carrying out product detection.Control strain Origami(DE3)/pBAD43/pETDUET-1 test of fermenting in the same way.Experiment in triplicate.Wherein, the formula of 100mM phosphate buffered saline buffer (pH7.4) is as follows: K
2hPO
43H
2o18.3g, KH
2pO
42.7g, is settled in 1L deionized water.
Two, target product S-1, the detection of 2-propylene glycol
1, high performance liquid chromatography detects
S-1, the preparation of 2-propylene glycol standard solution: get S-1,2-propylene glycol standard substance (sigma, catalog number (Cat.No.) 540250), are mixed with the solution of proper concn with deionized water.
The preparation of testing sample solution: the fermented liquid that step 1 is obtained, 12000rpm, gets supernatant after 2min is centrifugal, with 0.22 μ m membrane filtration, obtains the testing sample solution of the machine for the treatment of.
By S-1,2-propylene glycol standard solution and testing sample solution all carry out HPLC detection according to following condition: chromatographic column is organic acid post 300mm * 7.8mm Aminex HPX-87H (Bio-Rad); Moving phase is 6mM H
2sO
4; Flow velocity is 0.5ml/min; Applied sample amount is 10 microlitres; 55 ℃ of column temperatures; Differential refraction detector.According to S-1, in the fermented liquid that the HPLC collection of illustrative plates determination step one of 2-propylene glycol standard solution and testing sample obtains, whether contain S-1,2-propylene glycol.
Experiment arranges control strain Origami(DE3 simultaneously)/pBAD43/pETDUET-1 is in contrast.
Result shows, S-1, the HPLC collection of illustrative plates of 2-propylene glycol standard substance as in Fig. 2 a) as shown in, as can be seen from the figure, S-1, the appearance time of 2-propylene glycol standard substance is 19.722min.The HPLC collection of illustrative plates of the ferment filtrate of recombinant bacterial strain Origami(DE3)/pBAD-pct/pETDUET-pm is as b in Fig. 2) as shown in, with S-1, also there is target peak in the position that 2-propylene glycol standard substance retention time is consistent.Control strain Origami(DE3) this target peak not in the ferment filtrate of/pBAD43/pETDUET-1.According to above result, in the fermented liquid that preliminary judgement step 1 obtains, contain S-1,2-propylene glycol, Pfansteihl is to S-1, and the approach of 2-propylene glycol is got through.
2, Mass Spectrometric Identification
S-1, the preparation of 2-propylene glycol standard solution: get S-1,2-propylene glycol standard substance (sigma, catalog number (Cat.No.) 540250), are mixed with the solution of proper concn by ethyl acetate.
The preparation of testing sample solution: the fermented liquid that step 1 is obtained, 12000rpm, 10min, 4 ℃ are centrifugal, collect supernatant, regulate pH<2.0, after add the ethyl acetate of 0.5 times of volume, organic phase is collected in extraction.
By S-1,2-propylene glycol standard solution and testing sample solution (organic phase after ethyl acetate extraction), use respectively 0.22 micron of organic membrane filtration, carries out respectively GC/MS mensuration.Actual conditions is as follows: instrument model: the Trace ISQ gas chromatograph-mass spectrometer (gas phase: Trace1300) that ThermoFisher company produces.GC condition: chromatographic column: TG-WAXMS capillary column (30m * 0.25mm * 0.25 μ m).Carrier gas: helium, flow velocity 1ml/min.Temperature programming: 80 ℃ maintain 1min, rear 5 ℃/min is warming up to 200 ℃, maintains 5min.Input mode: split stream sampling, 220 ℃, splitting ratio 1:10,1 μ l sample introduction.Detector: EI source, 200 ℃ of mass ion source temperature, 250 ℃ of transmission line temperature, start mass spectrometric detection from 9.5min, mass scanning scope: 35-300m/z.
S-1,2-propylene glycol standard solution and testing sample solution (ethyl acetate extraction after organic phase) at the mass spectrum that goes out peak material of the same retention time of vapor detection respectively as shown in A in Fig. 3 and B, as can be seen from the figure, two mass spectrums match, and prove same substance.According to above result, in the fermented liquid that further determination step one obtains, contain S-1,2-propylene glycol, Pfansteihl is to S-1, and the approach of 2-propylene glycol is got through.
Claims (10)
1. prepare a S-1, the method for 2-propylene glycol, comprises with restructuring biomass cells and Pfansteihl or its salt is converted into S-1, the step of 2-propylene glycol; Described restructuring biomass cells is expressed to have and Pfansteihl or its salt is converted into S-1, the enzyme system of 2-propylene glycol function.
2. method according to claim 1, is characterized in that: described enzyme system comprises as follows (a1)-(a3):
(a1) there is the enzyme 1 that Pfansteihl or its salt is converted into L-lactoyl coenzyme A function;
(a2) there is the enzyme 2 that described L-lactoyl coenzyme A is converted into L-lactic aldehyde function;
(a3) have described L-lactic aldehyde is converted into S-1, the enzyme 3 of 2-propylene glycol function.
3. method according to claim 2, is characterized in that: described enzyme 1 is propionyl-coa transferase;
Described enzyme 2 is coenzyme A dependent form succinic semialdehyde dehydrogenase;
Described enzyme 3 is 3-hydroxymalonate dehydrogenase.
4. method according to claim 3, is characterized in that: the aminoacid sequence of described propionyl-coa transferase is as shown in sequence in sequence table 1;
The aminoacid sequence of described coenzyme A dependent form succinic semialdehyde dehydrogenase is as shown in sequence in sequence table 2;
The aminoacid sequence of described 3-hydroxymalonate dehydrogenase is as shown in sequence in sequence table 3.
5. according to arbitrary described method in claim 1-4, it is characterized in that: described restructuring biomass cells is following encoding gene to be imported to the restructuring biomass cells of the described enzyme system of expression obtaining after object biomass cells:
The encoding gene of propionyl-coa transferase as described in as shown in sequence in sequence table 4;
The encoding gene of coenzyme A dependent form succinic semialdehyde dehydrogenase as described in as shown in sequence in sequence table 5;
The encoding gene of 3-hydroxymalonate dehydrogenase as described in as shown in sequence in sequence table 6.
6. according to arbitrary described method in claim 1-5, it is characterized in that: described biomass cells is microorganism cells, zooblast or vegetable cell.
7. method according to claim 6, is characterized in that: described biomass cells is intestinal bacteria.
8. according to arbitrary described method in claim 1-7, it is characterized in that: described restructuring biomass cells is the recombination bacillus coli 1 described in claim 9.
9. following (I) or biomaterial (II) or (III):
(I) all or part of in following DNA fragmentation:
(b1) nucleotides sequence is classified the DNA fragmentation shown in sequence 4 in sequence table as;
(b2) nucleotides sequence is classified the DNA fragmentation shown in sequence 5 in sequence table as;
(b3) nucleotides sequence is classified the DNA fragmentation shown in sequence 6 in sequence table as;
(II) recombinant plasmid A and/or recombinant plasmid B:
Described recombinant plasmid A is for carrying and express the recombinant expression vector of DNA fragmentation described in (b1); Described recombinant plasmid B for carry and express DNA fragmentation described in (b2) and (b3) described in the recombinant expression vector of DNA fragmentation.
(III) recombination bacillus coli 1 or/and recombination bacillus coli 2 or/and recombination bacillus coli 3:
Described recombination bacillus coli 1 is for carrying recombinant plasmid A described in (II) and described recombinant plasmid B, and express DNA fragmentation described in DNA fragmentation, (b2) described in (b1) and (b3) described in the intestinal bacteria of DNA fragmentation;
Described recombination bacillus coli 2 is for carrying recombinant plasmid A described in (II), and the intestinal bacteria of DNA fragmentation described in expression (b1);
Described recombination bacillus coli 3 is for carrying recombinant plasmid B described in (II), and expresses b2) described in DNA fragmentation and (b3) described in the intestinal bacteria of DNA fragmentation.
10. biomaterial claimed in claim 9 is utilizing Pfansteihl or its salt to synthesize S-1, the application in 2-propylene glycol.
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Cited By (3)
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| CN104109651A (en) * | 2014-07-23 | 2014-10-22 | 中国科学院微生物研究所 | Recombinant Escherichia coli for synthesizing S-1,2-propanediol from L-lactic acid and construction method thereof |
| CN105651914A (en) * | 2014-12-02 | 2016-06-08 | 深圳翰宇药业股份有限公司 | Method for direct determination of chiral eutectic solvent in SGLT2 inhibitor eutectic |
| CN107746856A (en) * | 2017-10-19 | 2018-03-02 | 中国科学院天津工业生物技术研究所 | Produce construction method and the application of the Corynebacterium glutamicum recombinant bacterial strain of the rare sugar of L |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104109651A (en) * | 2014-07-23 | 2014-10-22 | 中国科学院微生物研究所 | Recombinant Escherichia coli for synthesizing S-1,2-propanediol from L-lactic acid and construction method thereof |
| CN105651914A (en) * | 2014-12-02 | 2016-06-08 | 深圳翰宇药业股份有限公司 | Method for direct determination of chiral eutectic solvent in SGLT2 inhibitor eutectic |
| CN107746856A (en) * | 2017-10-19 | 2018-03-02 | 中国科学院天津工业生物技术研究所 | Produce construction method and the application of the Corynebacterium glutamicum recombinant bacterial strain of the rare sugar of L |
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